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Article

Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China

by
Feng-Ming Yu
1,2,3,
Kandawatte Wedaralalage Thilini Chethana
1,2,
De-Ping Wei
1,2,
Jian-Wei Liu
1,2,4,
Qi Zhao
3,5,6,7,
Song-Ming Tang
1,2,8,
Lu Li
3,5 and
Kevin David Hyde
1,2,3,*
1
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
2
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
4
The Germplasm Bank of Wild Species, Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
5
Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming 650201, China
6
Institute of Applied Fungi, Southwest Forestry University, Kunming 650224, China
7
School of Chemical Engineering, Guizhou Institute of Technology, Guiyang 550003, China
8
Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Science, Kunming 650205, China
*
Author to whom correspondence should be addressed.
Pathogens 2021, 10(11), 1389; https://doi.org/10.3390/pathogens10111389
Submission received: 14 September 2021 / Revised: 13 October 2021 / Accepted: 20 October 2021 / Published: 27 October 2021
(This article belongs to the Section Fungal Pathogens)
Browse Figures
Versions Notes

Abstract

:
Tolypocladium, a diverse genus of fungicolous fungi belonging to Ophiocordycipitaceae, includes saprotrophic soil inhabitants, plant endophytes and pathogens of insects, nematodes, rotifers, and parasites of truffle-like fungi. Here, we review the research progress achieved for Tolypocladium regarding its taxonomy, species diversity, geographic distribution, host affiliations and ecological diversity. Furthermore, an undescribed taxon from China was established using morphology and multi-gene phylogeny. Tolypocladium inusitaticapitatum is introduced as a new species parasitizing ectomycorrhizal Elaphomyces species. It is diagnosed by its irregularly enlarged fertile heads and lemon, yellow-to-dark-brown, smooth and nearly cylindrical stipe. Phylogenetic analyses based on SSU, LSU, ITS, TEF1-α and RPB2 sequence data showed T. inusitaticapitatum to be an independent lineage separated from T. flavonigrum in the clade comprising T. capitatum, T. fractum and T. longisegmentatum. A key for identifying the sexual Tolypocladium species is also provided.

1. Introduction

Fungal species establish antagonistic to mutualistic associations with numerous prokaryotes and eukaryotes, including bacteria, algae, animals, plants and other fungi [1]. More than 1500 fungicolous taxa are widely distributed in aquatic and terrestrial ecosystems from tropical to polar regions [1]. Their hosts are ecologically diverse across the fungal kingdom. Truffle-like fungi are hypogeous and taxonomically distributed in Ascomycota and Basidiomycota [2]. Some truffle-like fungi were reported to be hosts of fungicolous species belonging to Absidia Tiegh., Battarrina (Sacc.) Clem. and Shear, Entoloma P. Kumm., Hypocrea Fr., Hypomyces (Fr.) Tul. and C. Tul., Hypoxylon Bull., Melanospora Corda, Sporothrix Hektoen and C.F. Perkins, and Tolypocladium W. Gam [1,3].
Tolypocladium W. Gams was established based on three soil-inhabiting asexual species: Tolypocladium cylindrosporum W. Gams, T. geodes W. Gams and T. inflatum W. Gams (the type species) [4]. Hodge and colleagues linked the asexual T. inflatum to the sexual species Cordyceps subsessilis Petch [5]. Subsequently, Sung and colleagues introduced the sexual genus Elaphocordyceps G.H. Sung and Spatafora and linked it to the asexual Tolypocladium and some species within Verticillium Nees based on multigene phylogeny [6]. Moreover, Sung and colleagues transferred the species of Cordyceps sensu lato that parasitize ectomycorrhizal Elaphomyces (18 species and two forma), cicada nymphs (C. inegoënsis Kobayasi, C. paradoxa Kobayasi, and C. toriharamontana Kobayasi) and beetle larvae (C. subsessilis) to Elaphocordyceps [6]. Chaunopycnis was established by Gams to accommodate Ch. alba, which resembles Tolypocladium in conidiogenesis [7]. Later, Quandt and colleagues synonymized Chaunopycnis and Elaphocordyceps under Tolypocladium, following the “One Fungus One Name” rule, as Tolypocladium is much more widely known, medicinally important and an older genus [4,6,7,8].
Most Tolypocladium species are Elaphomyces-attacking mycoparasites, except for few entomopathogens [9,10]. The evolution of host specificity and the dynamics of host jumping were investigated by several researchers using molecular data [6,8,11,12,13,14,15]. Nikoh and Fukatsu inferred that there was a shift from entomoparasitism to mycoparasitism during the evolution of the Cordyceps-like fungi [11]. However, with the addition of more gene regions and taxa, insect pathogens such as T. paradoxum and T. inflatum were found to be clustered with some parasites on truffles. The researchers explained that the ancestral ecology was a truffle parasitism, with multiple switches to insect pathogenicity [6,8,12]. Notably, the interspecific relationships of closely related Tolypocladium species are weakly supported and inconsistently resolved with different datasets [6,8,13,14]. To compensate for the shortage of limited loci, Quandt and colleagues performed genome-scale phylogenetic analyses based on two entomopathogens (T. ophioglossoides and T. capitatum) and two mycoparasites (T. inflatum and T. paradoxum) and demonstrated that truffle parasites form a monophyletic clade. They suggest that this lineage is derived as a result of a single ecological transition or host-jumping from insects to fungi [15].
A successful infection caused by fungal pathogens generally undergoes host recognition, attachment, and then infection and degradation, depending on the gene content, expression, or regulation [16]. Tolypocladium is recognized as an ideal candidate for investigating the mechanisms associated with host-jumping [15,16]. Quandt and colleagues researched the set of genes that are differentially regulated in Tolypocladium species during their first encounter with their hosts [16]. They found that PTH11-related G-protein-coupled receptors (GPCRs), predicted to be involved in host recognition, were up-regulated in T. ophioglossoides when grown on media containing insect cuticles [16]. Furthermore, a divergent chitinase and an adhesin gene, Mad1, were significantly up-regulated on media containing Elaphomyces [16]. According to the transcriptomic data, genes involved in redox reactions and transmembrane transport were the most overrepresented during T. ophioglossoides growth on Elaphomyces media. However, the genes involved in secondary metabolism may not be necessary for the parasitism of truffles as their products are only highly expressed during the growth on insect tissues [16].
To date, Tolypocladium comprises 41 species (Table 1) with a cosmopolitan distribution [2,17]. Some of them produce various secondary metabolites, such as cyclosporin, efrapeptins, ophiocordin and ophiosetin [18]. They have been widely used in biopharmaceuticals and biocontrol [18]. During an investigation of fungi in Yunnan Province, Southwest China, an undescribed Tolypocladium species was discovered on Elaphomyces sp. The present study aimed to (i) systematically review species diversity, hosts/habitat, geographical distribution and host affiliations of Tolypocladium species, (ii) broaden the knowledge of species diversity and host shifts in Tolypocladium species, (iii) refine the diagnostic characters of the interspecific classification of Tolypocladium in sexual morphs and provide a taxonomic key.

2. Results

2.1. Phylogenetic Placement

The combined SSU, LSU, ITS, TEF1-α and RPB2 sequence dataset comprised 35 species, containing 5384 nt (SSU: 1–1536, LSU: 1537–2441, ITS: 2442–3306, TEF1-α: 3307–4264, RPB2: 4265–5384) after the alignment (including gaps). Among them, 3731 bp (base pairs) were conserved, 378 variable, parsimony-uninformative, and 1275 parsimony-informative. The ML and BI analyses resulted in phylogenetic trees with a similar topology. The ML tree with a final log-likelihood of −27186.604 is shown in Figure 1. Specimens HKAS 112152 and HKAS 112153 clustered together and formed a distinct clade with strong support values (SH-aLRT = 100, UFB = 100 and BIPP = 1), indicating a conspecific relationship. These two specimens separated from other Tolypocladium species with SH-aLRT = 90.2 and BIPP = 0.98 support values. However, their LSU sequences showed an 11 bp difference (1.28%) across the 862 bp region, contributing to the different branch lengths in the phylogenetic tree. Based on the available molecular data for Tolypocladium species, some differences are known to occur due to intraspecific variations in the LSU sequences, ranging from 0.25 to 1.28% (Table 2).
Specimens Tolypocladium inusitaticapitatum (China), together with four Tolypocladium species occurring on Elaphomyces spp., i.e., T. capitatum (intercontinental distribution), T. flavonigrum (Thailand), T. fractum (USA) and T. longisegmentatum (intercontinental distribution), formed a monophyletic clade with weak support (SH-aLRT = 81.1, UFB = 82 and BIPP = 0.90. UFB values not shown in the ML tree). Tolypocladium inusitaticapitatum formed a separate clade sister to T. flavonigrum. However, the nucleotide comparison between T. inusitaticapitatum (holotype: HKAS 112152) and T. flavonigrum (holotype: BCC 66576) showed 154 bp (26.78%) differences across 575 bp ITS, 87 bp (9.83%) differences across 885 bp LSU, and 47 bp (4.99%) differences across 942 bp TEF1-α (including gaps), respectively. The phylogenetic evidence suggested that these two specimens represent new species.

2.2. Taxonomy

Tolypocladium W. Gams, Persoonia 6: 185 (1971); emended by Quandt and colleagues, IMA Fungus 5: 125 (2014).
Index Fungorum number: IF10242; Facesoffungi number: FoF 10425.
Synonyms: Chaunopycnis W. Gams, Persoonia 11: 75 (1980).
Elaphocordyceps G.H. Sung and Spatafora, Stud. Mycol. 57: 36 (2007).
Type species: Tolypocladium inflatum W. Gams 1971.
Morphological characterization: Sexual morph: Stromata arise directly from the host and are sometimes indirectly connected to the host through rhizomorph-like structures. They range from solitary to several and can be simple or branched. Stipe is fibrous to tough, rarely fleshy, dark-brownish to greenish with an olivaceous tint, rarely whitish, cylindrical and enlarges near the fertile part. The fertile part is clavate- to capitate-shaped and varies in color. Perithecia are partially to completely immersed, or superficial, or produced on a highly reduced stromatic pad, and ostiolate. Asci are unitunicate and long cylindrical with a thickened apical cap. Ascospores are filiform, approximately as long as asci, multi-septate, typically disarticulate into part-spores, and are occasionally non-disarticulating when mature (e.g., T. ramosum). Part-spores are hyaline, fusiform to cylindrical with round to truncate ends [6,8]. Asexual morph: They are Tolypocladium-, Chaunopycnis-, or Verticillium-like. Colonies are white, cottony and grow slowly on artificial media (e.g., potato dextrose agar, Czapek–Dox agar, malt extract agar, Sabouraud Glucose agar and water agar). Conidiophores usually are short and bear lateral or terminal phialides whorls. Phialides usually are swollen at the base and thin, often with bent necks. Conidia are globose to oval, one-celled, hyaline, smooth, and aggregative in small heads at the tips of the phialides [4,23].
Hosts and habits: Found in terrestrial and humid environments. Species of Tolypocladium parasitize hypogeous Elaphomyces (20 species including the novel species described in this study), cicada nymphs (4 species), beetle larvae (T. inflatum), pupa of the bagworm moth (T. fumosum), mosquito larvae (T. extinguens), and even bdelloid rotifers exposed to air (T. lignicola and T. trigonosporum). Their ascospores/conidia and mycelia survive in soil, or on various humus, rotting wood, plant tissues and surfaces, body surfaces of insects and mites, tissues of Cordyceps and lichens (Table 1).
Species diversity and distribution: Tolypocladium currently consists of 42 species (including the novel species described in this study) distributed worldwide [2,3,17]. Seventeen species were recorded from China (Table 1).

2.3. Description of the Novel Species

Tolypocladium inusitaticapitatum F.M. Yu, Q. Zhao and K.D. Hyde, sp. nov. Figure 2.
Index Fungorum: IF558123; Facesoffungi number: FoF 10407.
Typification: China, Yunnan Province, Lijiang City, Lijiang Alpine Botanic Garden, E100°10′58.07, N26°59′58.35, alt. 3338 m, 5 Oct 2019, Jian-Wei Liu (HKAS 112152, holotype).
Etymology: The specific epithet ‘inusitaticapitatum’ is derived from the combination of two Latin words, 1) adjective inusitata (strange, odd) and 2) noun capitatum (head), pointing to the fertile head, which is irregularly expanded.
GenBank accession numbers: ITS = MW 537735, LSU = MW 537718, SSU = MW 537733, TEF1-α = MW 507527, RPB2 = MW 507529.
Description: Asexual morph Stromata 9–11.5 cm high, solitary and simple, arising directly from the fruiting bodies of Elaphomyces sp. Stipe yellow at base, olive-brown to dark brown at the middle part, and yellowish brown at the terminal part. They are 7.5–11.5 cm long and 7–8.5 mm thick in the widest parts and nearly cylindrical, but the middle part is slightly thicker than the basal and upper parts. The fertile part developed from the terminal of the stipe, and is somewhat ellipsoidal, irregularly barrel-shaped, and sometimes slightly compressed, 1.5–2.0 cm × 1.5–2.0 cm. The surface is decorated with white ascospores released from the mature perithecia, which is olive yellow when immature, and olive to dark brown when mature. The outer layer becomes cracked and the olive internal texture is exposed. Structure of cortex of fertile part: composed of olive brown pseudoparenchymatous tissue and an ectal layer. Perithecia 580–720 μm × 180–270 μm (x = 650 μm × 220 μm, n = 10), crowded, entirely immersed, obovoid, ellipsoidal to pyriform. Ostioles papillate, and are visible (protruding up to 55 μm in high) or invisible, lined with periphyses. Asci is 410–510 μm × 10–15 μm (x = 461 μm × 13 μm, n = 20), hyaline, and long cylindrical, with a conspicuously thickened cap (measuring 6.5–7.5 μm × 6.0–7.0 μm). Ascospores are approximately as long as asci, and extremely easy to break into part-spores. Part-spores 20–32 μm × 3.0–4.5 μm (x = 25 μm × 3.6 μm, n = 20), hyaline, cylindrical with rounded ends. Asexual morph: Unknown.
Host and habitat: Directly arising from the fruiting bodies of hypogeous Elaphomyces sp. (Elaphomycetaceae, Eurotiales), in a humid and evergreen broad-leaved rainforest (Lijiang Alpine Botanic Garden), Lijiang, Yunnan Province, P.R. China. As serious degradation has occurred, truffle-like Elaphomyces sp. could not show any morphological evidence of taxonomic significance. Based on the ITS sequence dataset, the phylogenetic analyses showed that the host of T. inusitaticapitatum clustered together with Elaphomyces fuscus M. Shirakawa (Japan) and formed a sister group. However, there are sufficient molecular differences between the host from HKAS 112152 (ITS = MW 513695) and E. fuscus F-a170629 (ITS = LC 500967) to consider them as distinct species.
Known distribution: P.R. China (Yunnan).
Other specimen examined: CHINA, Yunnan, Lijiang, Lijiang Alpine Botanic Garden, alt. 3338 m, 5 October 2019, Jian-Wei Liu (HKAS 112153).
Notes: Based on the multi-gene phylogeny results, our specimens are closely related to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata directly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the perithecia entirely immersed in a well-differentiated valliforme-like structure [30]. However, T. inusitaticapitatum considerably differs from T. flavonigrum for the olive, yellowish-brown to dark brown fertile part, and is yellow to yellowish-brown at both ends of the stipe compared to the yellow–black to black stromata in T. flavonigrum. Tolypocladium inusitaticapitatum produces obovoid, ellipsoidal to pyriform perithecia, which are markedly distinguished from the elongate-ovoid perithecia produced by T. flavonigrum. Asci and part-spores of T. inusitaticapitatum (410–510 μm × 10–15 μm, 20–32 μm × 3.0–4.5 μm) are larger than those of T. flavonigrum ((318–)330–416(–482) μm × 7–8 μm, 2–5 μm × 1.5–2 μm) [30].
When comparing Tolypocladium inusitaticapitatum with its other phylogenetic relatives (T. capitatum, T. fractum and T. longisegmentatum), differences were found. Tolypocladium capitatum differs from T. inusitaticapitatum mainly due to its larger perithecia (900–1100 μm × 340–430 μm) and slimmer part-spores (2.5–3 μm wide) [10]. Tolypocladium fractum differs from T. inusitaticapitatum by having smaller stromata (1.5–2.5 cm long) and asci (300–480 μm × 5–6 μm) [10]. Tolypocladium longisegmentatum is distinguished from T. inusitaticapitatum by its longer stipe (13 cm long when fresh and up to 11 cm long when dried) and longer part-spores ((12–)40–65 μm) [20]. Morphologically, T. inusitaticapitatum is similar to T. intermedium for the yellow to dark brown stipe but differs in its smaller asci and shorter part-spores (main differences are outlined in Table 3). Regretfully, the molecular data of T. intermedium is not available in GenBank.

3. Discussion

Tolypocladium, a generalist genus, has been reported to have diverse lifestyles on a wide range of hosts and environments, including soil, insects, plants, lichens and hypogeal fungi [6,8]. The current pattern of host affiliation of Tolypocladium fungi is inferred to be an evolutionary product of intra- and inter-kingdom host shifts [57]. In the last two decades, researchers aimed to infer the evolution of host affiliation within the Tolypocladium, either using a handful of gene loci from dozens to hundreds of taxa, or using genome-scale data from fewer taxa [11,12,15,58]. To date, the studies on the host-jumping of Tolypocladium have been performed with multigene phylogeny (seven genes from 202 taxa of Hypocreales) [12] and genome-scale phylogeny (1350 genes from 20 taxa of Hypocreales) [15]. The multigene phylogenies supported three hypotheses for Tolypocladium, as follows: (1) the ancestral hosts were fungi (false truffles) [11,12,57,58]; (2) there were multiple switches to insect pathogenesis from a mycoparasitic ancestor [8,12,13]; (3) the endophytic lineage has arisen with the contact of plant hosts via mycorrhizal associations or plant-associated insects [12]. However, these conclusions, made from multigene phylogenies, conflict with those made from genome-scale phylogenies, which suggested a single ecological transition from insects to fungi within Tolypocladium [15]. Our phylogenic tree, inferred from five genes of 35 species (Figure 1), resulted in consistent conclusions, similar to those from previous multigene phylogenies. Similarly, we encountered several problems, such as phylogenetic conflicts among genetic data partitions and moderate to low support values for some important nodes [8,12,13]. Although whole-genome data provide insights that can further resolve the phylogenetic relationships of Tolypocladium [15,59,60], it is still unknown whether those conclusions will be limited by the few available species.
In this study, a novel Tolypocladium species occurring on Elaphomyces sp. is known from its sexual morph. A taxonomic key is also provided for 26 Tolypocladium species. The shape of the fertile part, the connection between the stipe and host, the structure of the cortex of the fertile part, size of part-spores and host affiliation are thought to be characteristic of taxonomic significance for interspecific identification [8,9,10]. However, there are 16 species whose sexual morphs are still unknown. In addition, the phylogenetic relationships among Tolypocladium species are very sensitive to taxa sampling and loci information [8,15]. Further studies should focus on obtaining more samples from different geographic regions and/or ecological niches, sequencing more markers and even genomic data, building a more robust phylogenetic relationship, and establishing their sexual–asexual morph connections. (Table 4).

4. Materials and Methods

4.1. Collections and Morphology

Tolypocladium specimens, including their underground host Elaphomyces sp., were collected in an evergreen broad-leaved forest in Lijiang Alpine Botanic Garden, Lijiang City, Yunnan Province, China. The specimens were examined as described in Senanayake and colleagues with the following modifications [61]. Colour codes were recorded following those of Kornerup and Wanscher [62]. Specimens were deposited at the Herbarium of Cryptogams Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China (HKAS, KUN).

4.2. DNA Extraction, PCR Amplification and Sequencing

The genomic DNA was extracted from the dried materials following the method described by Dissanayake and colleagues [63]. Fertile tissues from the parasitic fungi and the peridium of the host mushroom were used to extract DNA. Primer pairs ITS1F/ITS4 [64], LR0R/LR5 [65,66], PNS1/NS8 [64], TEF1-α 983F/TEF1-α 2218R [67] and fRPB2-5F/fRPB2-7R [68] were used for the amplification of the internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), the large subunit rDNA (LSU), the small subunit rDNA (SSU), the translation elongation factor 1-α (TEF1-α) gene and RNA polymerase II second-largest subunit (RPB2), respectively. PCR reaction was performed in a 25 μL reaction volume, comprising 12.5 μL Taq PCR Master Mix (Abmgood, Richmond, BC, Canada), 1 μL forward primer, 1 μL reverse primer, 2 μL DNA template and 8.5 μL ddH2O. For ITS, LSU, SSU and RPB2, PCR reaction conditions were as follows: 5 min at 94 °C, followed by 35 cycles of 40 s at 94 °C, 40 s at 53 °C and 1 min at 72 °C, and a final extension of 10 min at 72 °C. PCR reaction condition of TEF1-α was as follows: 5 min at 94 °C, followed by 35 cycles of 50 s at 94 °C, 40 s at 64 °C and 1 min at 72 °C, and a final extension of 10 min at 72 °C. The PCR products were visualized using agarose gel electrophoresis after staining with dyes (TS-GelRed Ver.2, Tsingke Biotechnology Co., Ltd., Beijing, China). Then, the products were sent for sequencing at Sangon Biotech Co. Ltd., Shanghai, China.

4.3. Sequence Alignment and Phylogenetic Analyses

Phylogenetic trees were constructed using the sequencing data of T. inusitaticapitatum and the allied reference sequences of closely related Ophiocordycipitaceae species obtained from the GenBank (Table 5). Aschersonia confluens (BCC 7961) and A. paraphysata (BCC 1467) of Clavicipitaceae were used as outgroup taxa. All sequences were assembled and aligned using MAFFT v 6.8 [69] and manually edited where necessary in BioEdit version 7.0.9 [70]. Individual alignments were compiled for SSU, LSU, ITS, TEF1-α and RPB2 genes. The optimal substitution model for each gene dataset was determined using MrModeltest 2.3 [71] under the Akaike information criterion (AIC). The results indicated that the GTR+I+G model was optimal for all the gene regions. Individual datasets were combined to assemble the combined dataset (gene order: SSU, LSU, ITS, TEF1-α and RPB2). The resulted combined dataset was deposited in the TreeBASE database (http://purl.org/phylo/treebase/phylows/study/TB2:S27887?x-access-code=746eddc746009259527edd3d4c69526b&format=html, accessed on 10 March 2021).
Maximum likelihood (ML) analysis was performed using IQ-Tree (http://iqtree.cibiv.univie.ac.at/, accessed on 20 May 2021) [72,73]. The substitution model options for each gene were auto-evaluated according to the provided partition file. Clade support for the ML analysis was assessed using an SH-aLRT test with 1000 replicates [74] and the ultrafast bootstrap (UFB) [75]. In the ML analyses, nodes with support values of SH-aLRT ≥ 80 and UFB ≥ 95 were considered well-supported, those with either SH-aLRT < 80 or UFB < 95 were considered weakly supported, and nodes with SH-aLRT < 80 and UFB < 95 were considered unsupported.
Bayesian Inference (BI) analysis was carried out in MrBayes v3.2.6 [76]. Gaps were treated as missing data. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 10,000,000 generations and were sampled at every 100th generation until the standard deviation of the split frequencies fell below 0.01 and ESS values > 200. Subsequently, phylogenetic trees were summarized and posterior probabilities (PP) were calculated using MCMC by discarding the first 25% generations as the burn-in phase [77]. Phylogenetic trees were viewed in FigTree v.1.4.4. Nodes with BI posterior probability (BIPP) > 0.90 were considered to be well supported.

Author Contributions

This study was initiated by F.-M.Y. and K.D.H. Samples were collected by J.-W.L. Morphological observation and description were done by F.-M.Y., K.D.H., K.W.T.C., D.-P.W., S.-M.T., J.-W.L. and L.L., and phylogeny analyses were done by F.-M.Y., K.W.T.C. and Q.Z. The manuscript was mainly drafted by F.-M.Y. with contributions from all other authors. All authors have read and agreed to the published version of the manuscript.

Funding

This research is supported by the National Natural Science Foundation of China, grant “Characterization of roots and their associated rhizosphere microbes in agroforestry systems: ecological restoration in high-phosphorus environment” (Grant No. 31861143002), “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion” (Grant No. RDG6130001), the Open Research Project “Cross-Cooperative Team” of the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences (Grant No. 292019312511043), Guizhou Science and Technology Planning Project (Guizhou Science and Technology Cooperation Support [2021] General 200) and the Joint Agricultural Program of Yunnan Province (No.2018FG001 032).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The obtained gene sequences were deposited in the NCBI GenBank database. The accession numbers of the obtained sequences are contained within the article and in Table 5.

Acknowledgments

The authors would like to express their sincere thanks to Ling-Sheng Zha (School of Life Sciences, Huaibei Normal University, Huaibei 235000, People’s Republic of China) for his help in finding literature and comments on this work; to Shaun Pennycook (Manaaki Whenua—Landcare Research, Private Bag 92170, Auckland 1072, New Zealand) for nomenclatural review; to Jennifer Luangsa-ard (National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand) for providing detailed information on Tolypocladium flavonigrum.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Maximum likelihood (ML) tree of Tolypocladium inusitaticapitatum and its allies within Ophiocordycipitaceae inferred from combined SSU, LSU, ITS, TEF1-α and RPB2 dataset. Bootstrap support values for ML ≥ 80 of SH-aLRT or 95 of UFB and posterior probability for BI ≥ 0.90 are indicated above the nodes and separated by ‘-/-/-’ (SH-aLRT/UFB/BIPP). Specimens of the current study are given in red. Type specimens are in bold and the superscript ‘ex’ indicates ex-type.
Figure 1. Maximum likelihood (ML) tree of Tolypocladium inusitaticapitatum and its allies within Ophiocordycipitaceae inferred from combined SSU, LSU, ITS, TEF1-α and RPB2 dataset. Bootstrap support values for ML ≥ 80 of SH-aLRT or 95 of UFB and posterior probability for BI ≥ 0.90 are indicated above the nodes and separated by ‘-/-/-’ (SH-aLRT/UFB/BIPP). Specimens of the current study are given in red. Type specimens are in bold and the superscript ‘ex’ indicates ex-type.
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Figure 2. Tolypocladium inusitaticapitatum (holotype: HKAS 112152). (a) Habitat; (b) Stromata arising from the fruiting bodies of Elaphomyces sp.; (c) Fertile heads; (d) Decomposed Elaphomyces sp.; (e) Ascospores released from mature perithecia (shown by a red arrow); (f) Vertical section of a fertile head; (g) Median section across the ostiole of the perithecium; (h) Vertical section across the cortex of a fertile head; (i–n) Asci with ascospores; (o) A thickened cap; (p,q) Part-spores. Bars: (b) = 10 cm; (c,d) = 2 cm; (e) = 2 mm; (f) = 500 μm; (g) = 50 μm; (h) = 100 μm; (i–n) = 250 μm; (o–q) = 20 μm.
Figure 2. Tolypocladium inusitaticapitatum (holotype: HKAS 112152). (a) Habitat; (b) Stromata arising from the fruiting bodies of Elaphomyces sp.; (c) Fertile heads; (d) Decomposed Elaphomyces sp.; (e) Ascospores released from mature perithecia (shown by a red arrow); (f) Vertical section of a fertile head; (g) Median section across the ostiole of the perithecium; (h) Vertical section across the cortex of a fertile head; (i–n) Asci with ascospores; (o) A thickened cap; (p,q) Part-spores. Bars: (b) = 10 cm; (c,d) = 2 cm; (e) = 2 mm; (f) = 500 μm; (g) = 50 μm; (h) = 100 μm; (i–n) = 250 μm; (o–q) = 20 μm.
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Table
Table 1. Species diversity, hosts/habitats and geographic distribution of Tolypocladium species.
Table 1. Species diversity, hosts/habitats and geographic distribution of Tolypocladium species.
Fungal NameHosts/Isolated FromKnown Distribution
T. albumSoil, sapwood of Hevea brasiliensisColombia, France, Scotland, Sri Lanka, Sweden, The Netherlands [7], Peru [12]
T. amazonenseSapwood of Hevea brasiliensis and H. guianensisPeru [12]
* T. capitatumElaphomyces granulatus, E. japonicus, Elaphomyces sp.Asia (China (Taiwan, Yunnan), Japan), Europe (France, Holland, Hungary), North America (Canada, U.S.A.) [9,10,19,20,21,22]
T. cylindrosporumSoil, sewage, peat, roots of Picea mariana; Plecia nearctica, larvae of Aedes sierrensis, larvae of Aedes australis, larvae and pupae of Lucilia sericata, Drosophila larvae (Diptera)Brazil, China, Czech, England, New Zealand, Nepal, The Netherlands, The North Island, U.S.A. [4,23,24,25,26,27]
* T. delicatistipitatumE. asahimontanusChina (Jiangxi) [28], Japan [10]
* T. dujiaolongaeCicada nymphsChina (Anhui, Fujian, Jiangsu, Jiangxi, Zhejiang) [29]
T. endophyticumLiving sapwood of Hevea brasiliensis and H. guianensisBrazil, Mexico, Peru [12]
T. extinguensLarvae of Arachnocampa luminosa (Diptera)New Zealand [24]
* T. fractumE. appalachiensisU.S.A. (Tennessee) [9]
* T. flavonigrumElaphomyces sp.Thailand [30]
* T. fumosumCocooned pupa of bagworm moth (Psychidae) buried among mossesPoland [31]
T. geodesSoilAustria, Canada, China, Denmark, England, The Netherlands [4,23,26]
* T. guangdongenseElaphomyces sp.China (Guangdong) [32]
* T. inegoenseCicada nymphs (e.g., Hyalessa maculaticollis)China (Fujian, Taiwan) [33], Japan [34], Korea [6]
* T. inflatumLarvae of Scarabaeidae (e.g., Aphodiinae, Rutelinae) (sexual morph); soil, humus, Picea glauca, roots of P. mariana, surface of Mycobates sp. (Acari, Mycobatidae), sclerotium of Ophiocordyceps gracilis (asexual morph)Sexual morph: Japan, U.S.A. (Tennessee, North Carolina, Michigan, New York, Washington) [5]; asexual morph: Austria, Canada, China, Nepal, Germany, U.S.A. [4,23,26,35]
* T. intermediumE. granulatus, E. subvariegatusJapan, U.S.A. (New York) [10,36]
* T. japonicumE. granulatus, E. japonicus, E. neoasperulusAustria, Japan [10], China (Guizhou, Taiwan) [28,37]
* T. jezoenseE. anthracinus, E. miyabeanus, E. nopporensisJapan [10]
T. lignicolaRotting wood (parasitic in bdelloid rotifers)Canada (Ontario) [38]
* T. longisegmentatumE. granulatus, E. japonicus, E. muricatus, Elaphomyces sp.Asia (China (Jilin), Japan), Europe (England, Germany, Holland), North America (Canada, Mexico, U.S.A.) [9,10,20,21,39]
T. microsporumSoilCanada, Germany, The Netherlands, U.S.A. [23]
* T. minazukienseElaphomyces sp.Japan [40]
* T. miomoteanumElaphomyces sp.Japan [40]
T. nubicolaSoilCanada (Alberta), China (Guizhou) [23,41]
* T. ophioglossoidesE. granulatus, E. japonicus, E. muricatus, E. shimizuensis, E. titibuensis, and Elaphomyces sp.Commonly in Asia (e.g., China (Guangxi, Jiangsu, Jiangxi, Jilin, Shandong, Sichuan, Taiwan, Yunnan), Japan, Korea), Europe and North America [9,10,42,43,44]
T. ovalisporumLichen Polycauliona regalisAntarctica (King George Island) [45]
* T. paradoxumCicada nymphs (e.g., Platypleura kaempferi, Graptopsaltria nigrofuscata)China (Hainan, Yunnan) [46], Japan, Koera [34,47]
T. pustulatumSoil, twigs in oak forest, and living leaf of Kalmia latifoliaMexico (Nuevo León), Spain (Cádiz), U.S.A. (New Jersey) [48]
* T. ramosumElaphomyces sp.China (Anhui, Fujian, Gansu, Guangdong) [44,49,50]
* T. rouxiiE. variegatusFrance [51]
T. sinenseStroma and sclerotium of Ophiocordyceps sinensisChina (Yunnan) [52]
* T. szemaoenseE. granulatusChina (Yunnan) [53]
* T. tenuisporumHost not found (probably Elaphomyces sp.)U.S.A. (Pennsylvania) [9]
T. terricolaSoilFinland [54]
* T. toriharamontanumCicada nymph (Auritibicen bihamatus)Japan [34]
T. trigonosporumRotting stump (parasitic on bdelloid rotifers)Canada (Nova Scotia) [55]
T. tropicaleSapwood and leaf tissue of Hevea brasiliensisMexico, Peru [12]
T. tundrenseSoilCanada (Northwest Territories) [23]
* T. valliformeE. granulatus, Elaphomyces sp.Canada (Ontario), U.S.A. (Carolina, New York, Virginia) [9]
* T. valvatistipitatumE. granulatus, E. neoasperulusJapan [10]
* T. virensElaphomyces sp.Japan [56]
* indicates sexual morphs (25 species).
Table
Table 2. Intraspecific base-pair differences in LSU genes among Tolypocladium species.
Table 2. Intraspecific base-pair differences in LSU genes among Tolypocladium species.
Locus522532855 Ratio
Species
T. albumCBS 393.89 #CCC 0.35% (3/870 bp)
GB5502TT-
Locus202123242527 Ratio
Species
T. inflatumOSC 71235 #AGAACA 0.76% (6/794 bp)
CBS 127302GA---C
Locus48434 Ratio
Species
T. ophioglossoidesCBS 100239 #CC 0.25% (2/816 bp)
NBRC 106330TT
Locus164382405433442479496524 Ratio
Species
T. paradoxumNBRC 106958 #TCGCCCTG 0.90% (8/891 bp)
NBRC 100945CTATTTCA
Locus83744518196110124204210402Ratio
Species
T. inusitaticapitatumHKAS 112152 #TTAAAATTAAG1.28% (11/862 bp)
HKAS 112153CCGGGGCCGGT
The locus numbers refer to the base-pair positions of the gene sequences, and “#” represents the reference sequences. Gaps are indicated with ‘-’.
Table
Table 3. Main differences between T. intermedium and T. inusitaticapitatum.
Table 3. Main differences between T. intermedium and T. inusitaticapitatum.
T. intermedium [10]T. inusitaticapitatum (This Study)
Fertile partDark reddish brownOlive brown, yellowish-brown to dark brown
StipeSlender, 6–8.5 cm long and 2–4 mm thick, middle part clearly expanded, surface with many longitudinal grooves, upper part squamuloseThicker, 7.5–11.5 cm long and 7–8.5 mm thick, middle part indistinctly expanded, surface smooth
Asci240–300 μm × 7–8 μm, caps about 5 μm in diameter410–510 μm × 10–15 μm, caps 6.5–7.5 μm × 6.2–7.0 μm
Part-sporesShort, 3–6 (commonly 4.5) μm × 1.5–2 μm, truncated at two ends (shape)Long, 20–32 μm × 3.0–4.5 μm, cylindrical with rounded ends
DistributionJapan, USAP.R. China (Yunnan)
Table
Table 4. Key to Sexual Morphs of Tolypocladium species.
Table 4. Key to Sexual Morphs of Tolypocladium species.
1. Host insects2
1′. Host hypogeous Elaphomyces spp.7
2. Host beetle or moth larvae3
2′. Host cicada nymphs4
3. Fertile part capitate, with stellate appearance; perithecia ovoid to pear-shaped, 740–760 × 444–558 μm T. fumosum
3′. Fertile part, strap-shaped pseudostalk; perithecia superficial, narrow flask-shaped, 1000–1500 × 330–440 μmT. inflatum
4. Stromata arising from underground mycelial membrane or strand; part-spores 3–5 × 1.5–2 μmT. paradoxum
4′. Stromata arising directly from host5
5. Fertile part elongated, obpyriform; part-spores 1.5–2–2.5 × 1.5–1.7 μm wideT. toriharamontanum
5′. Fertile part oblong or clavate 6
6. Perithecia superficial or apparently half-immersed, pyriform, 520–550 × 260–280 μm; part-spores 2.5–3 × 2 μmT. inegoense
6′. Perithecia wholly immersed, ampullaceous, (233–)520–740(–780) × (250–)300–330 (–360) μm; part-spores 3–5(–7.0) × 2–3 μm T. dujiaolongae
7. Stroma attached to host by rhizomorphs 8
7′. Stroma arising directly from the host12
8. Part-spores articulate, moniliform, 3–3.5 × 2–2.5 μm T. szemaoense
8′. Part-spores with truncate or rounded ends 9
9. Stroma capitate 10
9′. Stroma solitary or rarely caespitose 11
10. Perithecia small, 480–540 × 225–255 μm; part-spores large-sized, 18–28 × 3–5 μmT. delicatistipitatum
10′. Perithecia 770–800 × 350–430 μm; part-spores medium-sized, 8–11 × 1.5–2 μmT. miomoteanum
11. Perithecia oblong with long neck, 700–720 × 200–250 μm; part-spores long, 20–30(50) × 3–4.5 μmT. jezoense
11′. Perithecia ovoid, 550–600 × 200–300 μm; part-spores small short rod-shaped, 2.5–5 × 1.5–2 μmT. ophioglossoides
12. Perithecia superficial, ascospores nonfracturedT. ramosum
12′. Perithecia entirely embedded or ostiole slightly projecting13
13. Fertile part, cortex composed of pseudoparenchymatous peridial layer, and with an ectal layer 14
13′. Fertile part, cortex composed of pseudoparenchymatous peridial layer, but without ectal layer19
14. Stromata clavate; perithecia narrowly ovoid, 750–1000 × 250–300 μm; part-spores cylindric, 6–8 × 1–1.5 μm T. tenuisporum
14′. Stromata capitate15
15. Part-spores, larger-sized, more than 20 μm long 16
15′. Part-spores, less than 20 μm long17
16. Part-spores (12–)40–65 × (3–)4–5 μmT. longisegmentatum
16′. Part-spores 20–32 × 3.0–4.5 μmT. inusitaticapitatum
17. Part-spores, medium-sized, (13–)16(–21) × 2.5–3 μmT. rouxii
17′. Part-spores, small-sized, 2.5–6 μm long 18
18. Perithecia elongate-ovoid, (560–)567–697(–750) × (200–)206–248(–250) μm; part-spores 2–5 × 1.5–2 μmT. flavonigrum
18′. Perithecia ovoid, 450–540 μm × 230–260 μm; part-spores 3–6 (commonly 4.5) × 1.5–2 μmT. intermedium
19. Stromata clavate20
19′. Stromata capitate21
20. Perithecia small, 245–495 μm long, deeply embedded; asci short, 195–270 μm longT. guangdongense
20′. Perithecia 500–550 μm long, ostiola slightly projecting; asci 330–370 μm longT. japonicum
21. Perithecia large, more than 900 μm long22
21′. Perithecia medium-sized, 400–700 μm long 23
22. Perithecia ovoid, 900–1100 × 340–430 μm; part-spores cylindric or somewhat fusoid, 18–27 (commonly 24) × 2.5–3 μm T. capitatum
22′. Perithecia ampullaceous, 900–930 × 220–250 μm; part-spores fusoid, 16–18 × 3 μmT. minazukiense
23. Stipe slender, less than 1.0 mm thick 24
23′. Stipe thick, columnar, 1.0–6.0 mm thick25
24. Perithecia 500–600 × 250–350 μm; part-spores 2–5 × 1.5–2 μm T. fractum
24′. Perithecia 400 × 250 μm; part-spores 6 × 1.5 μmT. virens
25. Asci 10–12 μm wide; part-spores medium-sized, 7.5–16 × 2.5–3 μmT. valvatistipitatum
25′. Asci slender, 6–8 μm wide; part-spores small-sized, 3–8 × 2 μm T. valliforme
Table
Table 5. Voucher information and GenBank accession numbers for samples appearing in the Tolypocladium phylogenetic tree.
Table 5. Voucher information and GenBank accession numbers for samples appearing in the Tolypocladium phylogenetic tree.
TaxonStrain/Specimen VoucherGenBank Accession Numbers
ITS28S18STEF1-αRPB2
Aschersonia confluensBCC 7961JN049841DQ384947DQ372100DQ384976DQ452465
A. paraphysataBCC 1467 DQ377987DQ372090DQ384967DQ452463
Drechmeria gunniiOSC 76404JN049822AF339522AF339572AY489616DQ522426
D. sinensisCBS 567.95MH862540AF339545AF339594DQ522343DQ522443
D. zeospora exCBS 335.80MH861269AF339540AF339589EF469062EF469109
Ophiocordyceps gracilisEFCC 8572JN049851EF468811EF468956EF468751EF468912
O. heteropodaEFCC 10125JN049852EF468812EF468957EF468752EF468914
Paecilomyces lilacinusCBS 431.87AY624188EF468844 EF468791EF468940
Pa. lilacinus exCBS 284.36AY624189FR775484 EF468792EF468941
Perennicordyceps cuboideaNBRC 101740JN943331JN941417JN941724KF049684
Pe. cuboideaNBRC 100941JN943329JN941416JN941725
Pe. paracuboideaNBRC 101742JN943338JN941431JN941710KF049685KF049669
Pe. paracuboideaNBRC 100942JN943337JN941430JN941711AB972954AB972958
Pe. prolificaTNS-F-18481KF049659KF049631KF049612KF049686
Pe. prolificaTNS-F-18547KF049660KF049632KF049613KF049687KF049670
Polycephalomyces aurantiacusMFLU 17-1393MG136919MG136913MG136907MG136877MG136873
Po. aurantiacusMFLUCC 17 2113MG136916MG136910MG136904MG136875MG136870
Po. marginaliradiansMFLU 17-1582MG136920MG136914MG136908MG136878MG271931
Po. marginaliradiansMFLUCC 17-2276MG1369 21MG136915MG136909MG136879MG271930
Po. nipponicusNBRC 101406JN943301JN941388JN941753
Po. nipponicusBCC 1682KF049664KF049638KF049620KF049694MF416463
Po. yunnanensisYHCPY1005KF977848KF977848KF977848KF977850KF977854
Po. yunnanensisYHHPY1006KF977849KF977849KF977849KF977851KF977855
Tolypocladium amazonenseVPB179KF747267 KF747329
T. amazonense exMS308 KF747134KF747314KF747099
T. capitatumNBRC 106325 JN941402JN941739AB968598AB968559
T. capitatumNBRC 100997 JN941401JN941740AB968597AB968558
T. cylindrosporumARSEF 2920MG228381 MG228390MG228387
T. cylindrosporumYFCC 1805001MK984581MK984577MK984565MK984569MK984573
T. endophyticumMX535KF747260KF747153KF747322KF747117
T. flavonigrum exBCC 66576MN338090MN337287 MN338495
T. flavonigrumBCC 66578MN338091MN337288 MN338496
T. flavonigrumBCC66580 MN337289 MN338497
T. fractumOSC 110990 DQ518759DQ522545DQ522328DQ522425
T. fumosumWA18945KU925171KU985053
T. geodesCBS 126054MH864065MH875520
T. inflatumOSC 71235JN049844EF469077EF469124EF469061EF469108
T. inflatumCBS 127302MH864514MH875949
T. inusitaticapitatumHKAS 112152MW537735MW537718MW537733MW507527MW507529
T. inusitaticapitatumHKAS 112153MW537736MW537719MW537734MW507528MW507530
T. jezoensetxid94205AB027365AB027365AB027319
T. longisegmentatumOSC 110992 EF468816 EF468919
T. nubicolaCBS 568.84MH861780MH873478
T. ophioglossoidesCBS 100239KU382155KJ878874KJ878910KJ878958
T. ophioglossoidesNBRC 8992JN943316JN941405JN941736AB968601AB968562
T. ovalisporumCBS 700.92AB457006
T. paradoxumNBRC 106958JN943324JN941411JN941730AB968600AB968561
T. paradoxumNBRC 100945JN943323JN941410JN941731AB968599AB968560
T. pustulatumMRL GB6597AF389189AF389190
T. tropicaleMX338KF747259KF747149KF747318KF747113
T. tropicale exIQ214KF747254KF747125 KF747090
T. tundrenseCBS 569.84MH861781MH873479
T. valliformeDAOM 196368AY245640 AY245648
New sequencing data are displayed in bold. Specimens of the current study are given in red. Type specimens are in bold; superscript ‘ex’ indicates ex-type.
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Yu, F.-M.; Thilini Chethana, K.W.; Wei, D.-P.; Liu, J.-W.; Zhao, Q.; Tang, S.-M.; Li, L.; Hyde, K.D. Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China. Pathogens 2021, 10, 1389. https://doi.org/10.3390/pathogens10111389

AMA Style

Yu F-M, Thilini Chethana KW, Wei D-P, Liu J-W, Zhao Q, Tang S-M, Li L, Hyde KD. Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China. Pathogens. 2021; 10(11):1389. https://doi.org/10.3390/pathogens10111389

Chicago/Turabian Style

Yu, Feng-Ming, Kandawatte Wedaralalage Thilini Chethana, De-Ping Wei, Jian-Wei Liu, Qi Zhao, Song-Ming Tang, Lu Li, and Kevin David Hyde. 2021. "Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China" Pathogens 10, no. 11: 1389. https://doi.org/10.3390/pathogens10111389

APA Style

Yu, F. -M., Thilini Chethana, K. W., Wei, D. -P., Liu, J. -W., Zhao, Q., Tang, S. -M., Li, L., & Hyde, K. D. (2021). Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China. Pathogens, 10(11), 1389. https://doi.org/10.3390/pathogens10111389

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